Enzyme-Linked Immunosorbent Assays Using Novel Japanese Encephalitis Virus Antigen Improve the Accuracy of Clinical Diagnosis of Flavivirus Infections

Abstract

Japanese encephalitis virus (JEV), a member of the genusFlavivirus in the family Flaviviridae, is the leading cause of endemic/epidemic viral encephalitis in Asia, including India, Thailand, Vietnam, Singapore, the Philippines, Taiwan, China, Korea, and Japan (40). It is also one of several mosquito-borne flaviviruses, in addition to four serotypes of dengue virus (DENV-1 to -4), that have experienced emergence and/or reemergence throughout the world, especially in the tropical regions (22,24). Sequential infection by multiple cocirculating flaviviruses in the affected population confounds serodiagnosis (20), disease burden estimation (23), and the impact on pathogenesis (10).Flavivirus infections elicit protective antibody responses primarily against the envelope (E) glycoprotein (20). The E protein contains three structural and functional domains. E domain I (EDI) is an eight-stranded ␤-barrel; it contains two large insertion loops forming the elongate dimerization EDII and containing the highly conserved internal fusion peptide. EDIII has an immunoglobulin (Ig)-like structure and contains the primary receptor-binding motifs (16,29). Murine monoclonal antibody (MAb) studies have demonstrated that EDI contains predominately type-specific nonneutralizing (non-Nt) epitopes, EDII contains cross-reactive epitopes eliciting both Nt and non-Nt antibodies, and EDIII contains the majority of the type-specific Nt epitopes (6, 31-34, 37, 38).Diagnostic enzyme-linked immunosorbent assays (ELISAs) are common, relatively quick, and efficient assays for clinical diagnosis, traditionally requiring the use of suckling mouse brain-grown (SMB) antigen and more recently utilizing noninfectious recombinant virus-like particle (VLP) antigen. Studies have shown that VLP antigens have higher performance accuracy than SMB antigens when used in ELISA for diagnosing flaviviral infections (8,13,14,28). However, both SMB and VLP antigens contain wild-type (WT) E proteins that exhibit the same cross-reactive epitopes as the virus responsible for the infection. The amino acids located in the highly conserved E glycoprotein fusion peptide, in particular Gly104, Gly106, and Leu107, have been identified as important flavivirus crossreactive epitope determinants (6,7,37,39). Thus, it is possible to develop cross-reactivity-reduced antigens by introducing substitutions for amino acids within the fusion peptide, thereby improving virus-specific diagnostic assays (7, 39). Recently, fusion peptide mutant VLPs for both St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) demonstrated dramatic reductions in the observed cross-reactivity of immunoglobulin M capture (MAC) ELISA, producing more accurate differentiation of both current and past WNV and SLEV infections (30).Here, we present results from mutagenesis in the fusion peptide region of the JEV E protein to identify and ablate cross-reactive E protein epitopes and utilize these mutant VLPs as improved serodiagnostic antigens. The JEV G106K/ L107D (KD) VLP exhibited the most dramatic red...

Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

“…In the same manner, limited cross-reactivity to the JEV and WNV antigens was observed in few YF-17D-vaccinated human serum specimens (see Table S1 in the supplemental material). These observations are consistent with previous reports that antibodies to prM/E and NS1 proteins cross-react highly to flaviviruses within the same serocomplex, and poorly to those from different serocomplexes (24,27,29,44). In the United States, the recommended test protocol to distinguish WNV infections from St. Louis encephalitis virus (SLEV) infections involves the initial screening of specimens by MAC-ELISA, and sometimes in tandem with IgG ELISA, followed by PRNT to confirm positive MAC-ELISA results (45); this might have a turnaround time of about 2 weeks.…”

Identification of a novel infection-enhancing epitope on dengue prM using a dengue cross-reacting monoclonal antibody

“…Also, PL10 could successfully distinguish DENV serotypes from other flaviviruses in immunized mice sera. The high degree of antibody cross-reactivity among different flaviviruses has been a diagnostic challenge to distinguish various flaviviral infections, and this limitation is apparent for members of DENV serotypes [57,58]. It has been previously reported that prM-specific antibodies could be applied as a diagnostic marker to distinguish previous infection of DENV from JEV [22].…”

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

“…The patients' or negative-control sera were diluted 1:2,000 in PBS as previously suggested (19), premixed with VLP antigens, added immediately to wells, and incubated at 37°C for 60 min. A total of 50 l of prM/E antibody-depleted sera was transferred to the precoated anti-human IgM or IgG plates for MAC/GAC-ELISA as described above.…”